Maternal Embryonic Leucine Zipper Kinase (MELK), a Potential Therapeutic Target for Neuroblastoma

This article is featured in Highlights of This Issue, p. 495

Neuroblastoma is a clinically heterogeneous pediatric cancer. In a subset of patients, the tumors will undergo spontaneous remission without any treatment, and children classified as low or intermediate risk (1) are readily cured with surgery with or without adjuvant chemotherapy. However, outcome for patients classified as high risk remains poor, with survival rates of <50% despite intensive multimodality therapy (1). Furthermore, survivors of high-risk neuroblastoma are at risk for significant treatment-related late effects (2). Thus, there is an urgent need to identify neuroblastoma vulnerabilities that can be targeted with new therapies that will be more effective and less toxic than our current strategies.

Maternal embryonic leucine zipper kinase (MELK), a member of the AMP protein kinase family of serine/threonine kinases (3), activates multiple cellular pathways that drive oncogenic growth (4). High levels of MELK are detected in many types of adult solid tumors and leukemia, and increased levels of MELK expression correlate with clinically aggressive disease and poor survival. Furthermore, genomic or pharmacologic inhibition of MELK has been shown to suppress tumor growth in vitro and in preclinical adult cancer models (3, 5–8), indicating that this kinase is a therapeutic target. A number of studies have shown that MELK inhibition also increases sensitivity to radiation and chemotherapy in preclinical adult cancer models, suggesting that combination treatments may also be effective strategies (3, 9–11).

Although the mechanisms by which MELK mediates aggressive tumor growth are not completely understood, MELK has been shown to bind and phosphorylate Forkhead Box Protein M1 (FoxM1; ref. 12). The activated MELK–FoxM1 complex directly binds to the promoter region of enhancer of zeste homolog 2 (EZH2) gene and induces transcription (11). Upregulation of EZH2, a lysine methyltransferase that catalyzes trimethylation of histone 3 at lysine 27 (H3K27me3), leads to the transcriptional repression of differentiation genes and maintains stem-like properties of cells. MELK-mediated EZH2 activity has also been shown to confer resistance to radiation in several adult cancer models (10, 11).

OTS167 is a potent small-molecule inhibitor of MELK (13). Nanomolar concentrations of OTS167 efficiently inhibit the proliferation of cancer cells that express high levels of MELK in many types of adult solid tumors (5, 6, 8, 13, 14), as well as acute myeloid leukemia (AML; ref. 7) and multiple myeloma (15). The drug, however, has little effect on cancer cells with low MELK expression (8, 13) or normal cells (15–18). On the basis of these promising studies, several adult cancer phase I studies testing OTS167 are currently being conducted (19).

Little is known about the role of MELK in the pathogenesis of pediatric cancers. However, Guan and colleagues have reported that MELK expression is significantly correlated to poor overall survival in patients with neuroblastoma. These investigators also showed that MYCN regulates MELK expression, and that the growth of neuroblastoma xenografts was suppressed with MELK inhibition (18). In this study, we confirmed the prognostic relevance of the level of MELK expression in an expanded cohort of patients with neuroblastoma. We also analyzed the correlations between MELK expression, tumor cell phenotype, and response to OTS167 in 11 well-characterized neuroblastoma cell lines with or without MYCN amplification. In addition, we investigated the antitumor activity of OTS167 in preclinical neuroblastoma models, and conducted combination studies with OTS167 to test for synergy with radiation or chemotherapy.

Neuroblastoma cell lines SK-N-DZ, LA1-55n, SH-SY5Y, NMB, SK-N-BE2, LAN-5, SMS-KCNR, NBL-W-N, NBL-W-S, LA1-5s, and SHEP were grown at 5% CO₂ in RPMI1640 (Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mmol/L l-glutamine, and 1% penicillin/streptomycin. NBL-W-N and NBL-W-S were established in our laboratory (20, 21), SK-N-DZ was purchased from ATCC, SMS-KCNR was a kind gift from Dr. Carol Thiele (NIH), LA1-55n, LA1-5s, SK-N-BE2, SHEP, and SH-SY5Y were kind gifts from Dr. June Biedler (Memorial Sloan-Kettering). All cell lines were authenticated by short tandem repeat profiling and were identical to reference profiles. SK-N-DZ and SK-N-BE2 were authenticated at ATCC using the PowerPlex 18D System (Promega), authentication of LA1-55n, SMS-KCNR, NBL-W-N, NBL-W-S, LA1-5s, and SHEP was performed at The Johns Hopkins University Fragment Analysis Facility (Baltimore, MD) using the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems). All cell lines tested negative for Mycoplasma contamination using the MycoAlert Detection Assay (Lonza).

RNA was isolated using TRIzol reagent (Life Technologies), and concentration was determined using UV spectroscopy (DeNovix). Reverse transcription was performed using Superscript III (Life Technologies) according to the manufacturer's instructions. RNA qPCR reactions were set up with 1× Power SYBR Green Master Mix (Applied Biosystems) and 250 nmol/L forward and reverse primers in a 20 μL reaction in a 96-well format. Real-time fluorescence detection of PCR products was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems) with 1 cycle at 95°C for 10 minutes; 40 cycles of 95°C for 15 seconds and 60°C for 1 minutes. Premade qPCR primer set Hs.PT.58.40892603 targeting exons 10–11 of MELK were purchased from IDT. Primers for GAPDH, which was used as an endogenous control, were described previously (22). Relative quantities of mRNA expression were determined using the comparative ΔΔC_t method.

The effects of OTS167 (OncoTherapy Science) on neuroblastoma cell proliferation in vitro were analyzed using the CellTiter 96 AQueous Non-radioactive Proliferation Assay Kit (Promega). Cells were plated in triplicate wells in a 96-well plate and OTS167 was added 24 hours later at concentrations ranging from 0 to 1 μmol/L. After a 72-hour incubation, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added and absorbance measured using a Synergy 2 Microplate Reader (Bio-Tek Instruments). A nonlinear regression, sigmoidal four-parameter dose–response model was used to determine IC₅₀ by plotting log of inhibitor concentration versus relative survival using Prism software (GraphPad). For synergy experiments, SK-N-BE2 and SK-N-DZ cells were plated as above and camptothecin (Alfa Aesar) and OTS167 were added alone or in combination at concentrations from 0 to 2 μmol/L. After a 48-hour incubation, MTS was added and absorbance measured as above. Combination index (CI) was determined with CompuSyn Software using the Chou–Talalay method (23).

SK-N-DZ and SK-N-BE2 were irradiated using a Philips RT250 Maxitron X-ray irradiator at 1.5 Gy/minute and then plated at 5,000 or 2,000 cells per well, respectively, in 6-well plates containing complete medium with OTS167 or 0.5% DMSO vehicle control. Cultures were grown until colonies of >50 cells had formed in the unirradiated DMSO vehicle control wells, 10 days for SK-N-DZ and 7 days for SK-N-BE2. SK-N-BE2 colonies were stained and imaged by washing each well once with PBS, staining and fixing with 0.5% crystal violet in 6% glutaraldehyde for 30 minutes, washing twice with water, drying, and imaging. The less-adherent SK-N-DZ colonies were stained by adding thiazolyl blue tetrazolium bromide (MTT) directly to the growth media to a final concentration of 0.05% and incubating the plates for 4 hours at 37 °C, 5% CO₂ before imaging. Automatic colony enumeration was performed using a custom set of macros for NIH ImageJ.

For the drug treatment studies, SK-N-BE2 and NMB cells were treated with indicated concentrations of OTS167 and/or camptothecin for 24 hours. For the radiation experiments, SK-N-BE2 cells were treated with 0, 2, or 4 Gy IR using a Philips RT250 Maxitron X-ray irradiator at 250 KVp and 15 mA with 1.0 mm Cu filter in 30/15 × 15 cm² cone, set at 10 cm from the sample with full backscatter. Indicated concentrations of OTS167 were added immediately after irradiation and incubated with cells for 24 hours. Protein lysates were prepared by boiling cell pellets in buffer containing 50 mmol/L Tris-HCl pH 6.8, 2% SDS, and protease/phosphatase inhibitor cocktail (Sigma-Aldrich) for 5 minutes. Protein concentrations were determined with the BCA Protein Assay Reagent (Pierce). Ten micrograms of total protein were electrophoresed on Stain-Free 4–20% SDS-PAGE gradient gels (Bio-Rad) and transferred to nitrocellulose membranes (Amersham). Membranes were blocked in TBS (pH 7.4) with 0.1% Tween-20 and 5% nonfat dry milk. Mouse EZH2 (Active Motif), rabbit pRPA32 (S4/S8; Bethyl Laboratories), mouse RPA32 (Novus Biologicals), rabbit MYCN (Cell Signaling Technology), and mouse MYC (Santa Cruz Biotechnology) antibodies were used at a 1:1,000 dilution. Mouse alpha tubulin antibody (Fitzgerald, 10R-T130A) was used at a 1:2,000 dilution, and mouse hsc70 (Invitrogen) and mouse anti-MELK (OncoTherapy Science) antibodies were used at a 1:10,000 dilution. Appropriate secondary antibodies were used and blots were developed with Clarity Western ECL Substrate (Bio-Rad). Images were acquired on a BioRad ChemiDoc XRS+ imaging system and analyzed using Image Lab software. For quantitative image analyses all volumes were normalized by the total amount of protein in each sample determined by gel activation or by the levels of α-tubulin.

SK-N-BE2, NBL-W-N, and NBL-W-S cells were treated with OTS167 from 0 to 8 nmol/L for 24 hours, harvested and washed with cold PBS (pH 7.4) followed by Accumax (Millipore) at room temperature for 10 minutes. The cells were pelleted at 1,200 rpm for 5 minutes, washed with PBS, and 95% ethanol was added drop wise while vortexing, followed by 30 minutes incubation at −20°C. Before cytometric analysis cells were washed two more times with PBS and 1 × 10⁶ cells were resuspended in 0.5 mL of Propidium Iodide/RNase Staining Buffer (BD Biosciences). Cellular DNA content was then collected using a BD LSR II flow cytometer and analyzed using BD FACSDiva Software (BD Biosciences).

To test the antitumor activity of OTS167 in vivo, female nude mice (Harlan) were subcutaneously injected with 1 × 10⁷ SMS-KCNR neuroblastoma cells as described previously (22). Animals were randomized to treatment (n = 9) or control groups (n = 10) when tumors reached approximately 70 mm³ and were palpable. The treatment group received 10 mg/kg OTS167 and control animals received vehicle alone. OTS167 or vehicle were injected intraperitoneally (i.p.) twice weekly for 3 weeks, tumor size was measured using a caliper, mice were weighed at time of measurement; and all animals were sacrificed 3 days after the last treatment. Tumors were resected, weighed, and parts of the tissue were snap-frozen or fixed in formaldehyde and paraffin-embedded for pathologic evaluation. Tissue sections were stained with hematoxylin and eosin (H&E), Masson trichrome, or anti-Ki67 antibody at the Pathology Core Facility at the University of Chicago (Chicago, IL). For the minimal residual disease (MRD) model, when tumors reached 70 mm³ mice were treated with 150 mg/kg cyclophosphamide (Baxter Healthcare) i.p. every other day for a total of six doses. Tumors were allowed to regress completely and the mice were randomized 5 days later to receive OTS167 (n = 8) or vehicle control (n = 8) 2×/week as above and treatment continued until tumor size reached 2,000 mm³. Animals were sacrificed and tumors removed as above. For injections, stock solutions of OTS167 were made up in DMSO and diluted in sterile 5% dextrose; cyclophosphamide was diluted in sterile water. All animal studies were conducted under protocol #71829 approved by the University of Chicago Institutional Animal Care and Use Committee (Chicago, IL).

Publicly available microarray gene expression data for the Kocak dataset (709 patients, E-MTAB-1781; ref. 24) and Versteeg dataset (88 patients, GSE16254; ref. 25) were downloaded from EMBL and GEO, respectively. Raw microarray data were normalized within each cohort using the robust multi-array analysis algorithm to account for batch effects (26). MELK expression for downstream analysis was extracted using the A_23_P94422 probe for the Kocak dataset and the 204825_at probe for the Versteeg dataset. EZH2 expression was quantified using the A_23_P259641 probe from the Kocak dataset. Differences in MELK expression between binary groups were assessed by pairwise Mann–Whitney U and multiple groups by the Kruskal–Wallis method.

Patients were categorized as having high or low expression of MELK using a sliding window to include at least 10% of patients similar to prior reports (27). The log-rank test was used to calculate the P value of the survival difference between high- and low-expression groups in each window. Multiple testing correction was done using the q value method (28) to account for the number of sliding windows. The most significant q value for each analysis is reported.

All in vitro experiments were repeated at least three times in triplicate and SDs were calculated. All animal studies had at least 5 mice per group and mean values of the tumor volumes, weights, and vessel densities were compared. All quantitative values obtained in the experiments were evaluated using paired Student t test. A P value of 0.05 was required to ascertain statistical significance. One-way ANOVA was used to calculate differences in MELK and EZH2 protein levels across treatments with increasing concentrations of OTS167.

To investigate whether high levels of MELK expression are associated with clinically aggressive neuroblastoma and poor outcome, we analyzed publicly available clinical and expression data in the Kocak dataset with 709 patients (E-MTAB-1781; Fig. 1A, C, E, and G), and the Versteeg cohort with 88 patients (GSE16254; Fig. 1B, D, and F). Survival was inferior for patients with high versus low levels of MELK expression in both cohorts (q values 2.1e⁻¹⁴ and 2.4e⁻³, respectively). High expression of MELK was also significantly associated with advanced stage of disease (P < 2.0e⁻¹⁶) and MYCN amplification (P = 2.2e⁻¹⁶). Although risk group information was not available for the 88-patient cohort, increased levels of MELK expression were correlated with high-risk disease in the 709-patient cohort with P = 2.2e⁻¹⁶ (Fig. 1G). However, within the high-risk cohort, the level of MELK expression was not statistically significantly associated with outcome in the subset of 254 high-risk patients in the Kocak dataset after multiple testing correction.

We next measured MELK mRNA and protein expression in 11 neuroblastoma cell lines by qPCR and Western blot analysis. MELK was detected in all of the cell lines, although the levels of expression varied (Fig. 2). High levels of MELK protein were observed in neuronal N-type cell lines. N-type cells express neuronal markers of differentiation, commonly have neurite extension when cultured on plastic, generally express high levels of MYCN, and form tumors in nude mice (29). We show that high levels of MYCN are expressed in all of the N-type cell lines we analyzed with the exception of SH-SY5Y, which expresses high levels of MYC (Supplementary Fig. S1). In contrast, low levels of MELK were detected in the S-type cell lines. These substrate-adherent cells, do not express neuronal markers but may express Glial Fibrillary Acidic Protein, and generally are not tumorigenic in vivo (29). Although MELK protein levels were higher in the tumorigenic N-type cell lines, MELK mRNA levels were more variable, suggesting that MELK may be additionally regulated at the level of translation and/or protein stability.

The effects of OTS167 on cell growth were evaluated in 11 neuroblastoma cell lines. The IC₅₀ was less than 9 nmol/L for all tested N-type tumorigenic cell lines (Fig. 3A and B). In contrast, the nontumorigenic S-type cells had low levels of MELK expression and were more resistant to OTS167 with IC₅₀ ranging from 25 to 44 nmol/L. Thus, similar to the adult cancers, low nanomolar concentrations of OTS167 inhibited proliferation of neuroblastoma cells with high MELK expression.

We also measured the effects of OTS167 on cell cycle in the N-type SK-N-BE2 cell line and N/S-type pair NBL-W-N and NBL-W-S. For this analysis, cells were exposed to OTS167 for 24 hours at concentrations below the IC₅₀ to prevent cytotoxicity and cell death. In both N-type cell lines, SK-N-BE2 and NBL-W-N, a decrease in the number of cells in G₁-phase and increase in the number of cells in S-phase was observed, which reached statistical significance at 8 nmol/L concentrations of OTS167. We also observed an increased number of cells in sub-G₁–phase at this concentration of OTS167, although statistical significance was not achieved (Fig. 3C). Although OTS167 induced similar changes in the cell cycle in S-type NBL-W-S cells, statistical significance was not reached and no increase in the number of cells in Sub-G₁–phase was observed. Thus, similar to previous studies in AML cells (7), the progression of mitosis is inhibited by OTS167 in neuroblastoma cells.

Intraperitoneal administration of 10 mg/kg of OTS167 twice per week also significantly inhibited the growth of neuroblastoma xenografts comprised of N-type SMS-KCNR cells (Fig. 4A). Animals were sacrificed after 3 weeks of treatment, and the average weight of the OTS167-treated tumors was significantly decreased compared with controls (0.45 ± 0.21 g vs. 2.00 ± 0.57 g; P < 0.05; Fig. 4B and C). Histologic examination of H&E-stained sections revealed large areas of hemorrhage in the control tumors, whereas little to no hemorrhage was detected in the tumors treated with OTS167 (Fig. 4D). Masson trichrome staining showed large areas of interstitial stroma with abundant depositions of extracellular matrix (ECM) in treated tumors, while control tumors where comprised almost exclusively of neoplastic cells (Fig. 4E). To detect proliferating neoplastic cells, xenograft sections were stained for Ki67. No difference in the intensity of staining was observed between control and treated sections (Fig. 4F).

To investigate the activity of OTS167 in the setting of MRD, we treated mice with this MELK inhibitor after chemotherapy-induced tumor regression. Mice with palpable neuroblastoma xenografts first were treated with cyclophosphamide. Following tumor regression, the mice were randomized to receive either 10 mg/kg of OTS167 or control vehicle, twice per week as above until tumor size reached 2,000 mm³. Although all mice treated with cyclophosphamide followed by OTS167 or vehicle eventually developed recurrent tumors, prolonged survival was seen in the cohort treated with OTS167 (104 ± 10 days vs. 80 ± 12 days; P = 0.01; Fig. 4G). Furthermore, the average tumor volume was significantly decreased in the animals treated with OTS167 from day 65 until day 72 (P < 0.05; Fig. 4H).

To investigate whether OTS167 directly modifies MELK expression in neuroblastoma, we measured MELK protein levels by Western blot analysis after 24 hours of treatment. Comparison of control versus treated cells, demonstrated that OTS167 statistically significantly decreased the quantity of MELK protein in SK-N-BE2, NMB, and SMS-KCNR neuroblastoma cell lines (P = 3.18e⁻⁰⁶, 0.000481, and 0.00902, respectively; Fig. 5A and B). We next evaluated the effects of OTS167 on EZH2, a known MELK target (11), by Western blot analysis, and found that EZH2 protein levels were also significantly decreased in SK-N-BE2, NMB, and SMS-KCNR cells with treatment (P = 2.55e⁻⁰⁵, 2.53e⁻⁰⁹, and 0.00587, respectively; Fig. 5A–C). Analysis of the Kocak expression data shows that the levels of MELK and EZH2 mRNA are highly correlated in primary tumors (Pearson r = 0.68; Fig. 5D), further supporting a role for MELK in the regulation of EZH2 in neuroblastoma. Interestingly, OTS167 treatment did not result in a decrease in MELK and EZH2 transcript levels in SK-N-BE2 cells (Supplementary Fig. S2B). Thus, OTS167-mediated posttranslational modifications may regulate MELK and EZH2 protein in neuroblastoma.

MELK inhibition has been reported to increase sensitivity to chemotherapy and radiation in adult cancers (3, 9–11). To investigate whether sensitivity to the DNA-damaging agent camptothecin is also increased in neuroblastoma cells when MELK is inhibited with OTS167, we tested both drugs for synergy using a constant dilution ratio to determine drug CI (23). With this method a CI > 1 indicates there is drug antagonism; a CI = 1 indicates the combination therapy is additive; and a CI < 1 indicates there is a synergistic interaction between the drugs. We analyzed the CI for OTS167 and camptothecin in SK-N-DZ and SK-N-BE2 cells and found that in both cell lines the drugs were synergistic at concentrations ranging from 3.125 nmol/L to 250 nmol/L (Fig. 6A). To investigate whether the sensitivity to radiotherapy is also increased by OTS167, clonogenic survival assays were performed, which demonstrated that OTS167 and radiation exerted supra-additive lethal effects in both neuroblastoma cell lines (Fig. 6B).

To investigate the mechanisms by which OTS167 treatment enhances sensitivity to camptothecin, we measured phosphorylation of serine 4 and 8 of Replication Protein A2 (RPA32), a marker of collapsed replication forks, in neuroblastoma cells treated with camptothecin alone or in combination with OTS167. Phosphorylation of the 32 kDa subunit of RPA32 was increased in neuroblastoma cells following treatment with camptothecin, indicative of the expected increase in formation of collapsed replication forks. Cotreatment of neuroblastoma cells with camptothecin plus OTS167 resulted in a reduction in collapsed replication forks, and it significantly decreased RPA32 phosphorylation with 10–50 nmol/L concentrations of OTS167 (P < 0.05; Fig. 6C). Similar results were observed in NBL-W-N and NBL-W-S cells (Supplementary Fig. S3). In experiments using radiation in place of camptothecin, treatment with 10 nmol/L and 50 nmol/L OTS167 also significantly reduced RPA32 phosphorylation in a dose-dependent manner (P < 0.05; Fig. 6D). These results suggest that MELK inhibition with OTS167 attenuates the processing of stressed replication forks into frank double-stranded breaks, thereby interfering with the normal processing of these genotoxic lesions, which in turn sensitizes neuroblastoma cells to radiation and camptothecin.

MELK is known to promote oncogenic growth in common adult malignancies, but its role in neuroblastoma pathogenesis remains largely unknown. In this study, we analyzed publicly available clinical and expression data and demonstrated that high levels of MELK expression in primary neuroblastomas were significantly associated with high-risk disease and poor outcome. We also found that MELK expression associates with the phenotype of neuroblastoma cells, with high levels of MELK detected in N-type neuroblastoma cell lines that are capable of forming tumors in nude mice, whereas lower levels were present in S-type cell lines that are not tumorigenic. Pharmacologic inhibition of MELK with OTS167 decreased the growth of neuroblastoma cells. Similar to adult cancer studies, we found that the sensitivity to OTS167 was increased in cell lines with high MELK expression. We also show that OTS167 suppressed the growth of neuroblastoma xenografts. Taken together, these results indicate that MELK may be a clinically relevant therapeutic target in neuroblastoma, and that the level of MELK expression may serve as a biomarker for response to OTS167.

We also tested OTS167 in a neuroblastoma mouse model of MRD. In patients with neuroblastoma, MRD is not detected by standard tumor evaluation tests and commonly persists following consolidation with myeloablative chemotherapy and stem cell transplant, eventually leading to relapse. In an effort to eliminate refractory microscopic disease, postconsolidation therapy is now part of standard treatment of high-risk neuroblastoma, and survival has been improved following the incorporation of isotretinoin and immunotherapy in this setting (30). There is increasing evidence that neuroblastoma stem cells or tumor-initiating cells are resistant to therapy and persist following treatment. Because MELK is important in the maintenance of tumor stem cells (9, 12, 31), we hypothesized that OTS167 may be effective in the setting of MRD. Our studies show that OTS167 treatment delayed relapse and prolonged survival of the animals with MRD following treatment with cyclophosphamide. However, tumor recurrence was observed in all of the mice, indicating that OTS167 alone was not sufficient to eliminate the residual neuroblastoma cells.

Our results confirm and expand the studies recently published by Guan and colleagues showing significant associations between high levels of MELK expression and poor survival in patients with neuroblastoma (18). These investigators also demonstrated that MYCN directly regulates MELK transcription, and they reported that the levels of MELK expression were statistically significantly higher in tumors with MYCN amplification. Although there was overlap of the datasets analyzed in the two studies, we evaluated an extended Kocak dataset containing expression data and clinical information for 709 patients and also analyzed an independent Versteeg dataset with 88 patients. We detected significantly higher levels of MELK in high-risk patients compared with the non-high-risk cohort. Among the high-risk subset of 254 patients, there was a trend associating inferior event-free survival with increased levels of MELK expression, although statistical significance was not met. In concert with our results, Guan and colleagues also demonstrated that OTS167 significantly inhibited neuroblastoma cell proliferation, blocked cell-cycle progression, and suppressed the growth of neuroblastoma xenografts. While many of the neuroblastoma cell lines evaluated in their study were different than the ones used in our study, SH-SY5Y was tested in both studies. In both studies, the growth of this MYCN–non-amplified cell line was suppressed with MELK inhibition, suggesting that this treatment strategy may be effective in neuroblastoma tumors with or without MYCN amplification. In the study by Guan and colleagues, 208 nmol/L of OTS167 was required to achieve the IC₅₀, while in our study the IC₅₀ for SH-SY5Y was 2.2 nmol/L. The reason for these disparities in OTS167 sensitivity are not clear, but may be related to differences in the drugs, which were obtained from different manufacturers, or experimental conditions of cell culture.

The mechanisms by which MELK promotes cancer growth remain largely unknown. Studies in glioma have shown that MELK phosphorylates and forms a complex with FoxM1, a transcription factor which functions as an oncogene and regulator of stem-like properties (12). This complex activates EZH2, a lysine methyltransferase, which catalyzes trimethylation of histone 3 at lysine 27, leading to inhibited transcription of genes involved in differentiation (11). Our studies suggest that MELK-dependent EZH2 signaling also occurs in neuroblastoma, as OTS167 treatment downregulated the protein levels of both MELK and EZH2. However, MELK and EZH2 RNA levels were not decreased in neuroblastoma cells with OTS167 treatment, indicating that the changes in MELK and EZH2 protein observed in neuroblastoma cells treated with OTS167 were not transcriptionally regulated. Rather, OTS167 may mediate posttranslational modifications that result in the downregulated expression of MELK and EZH2 protein in neuroblastoma. Indeed, in breast cancer, another small-molecule inhibitor MELK-T1 has been shown to inhibit MELK autophosphorylation, leading to proteasomal degradation (32). In medulloblastoma MELK directly binds and phosphorylates EZH2 (33). Although MYCN has been shown to regulate EZH2 (34, 35), significant correlations between MYCN and EZH2 expression in the neuroblastoma cell lines we analyzed were not detected. We also did not detect significant associations between MYCN and EZH2 expression the in primary tumors analyzed in the R2 database (http://r2.amc.nl). Further studies will be required to clarify the interactions between MYCN, EZH2, and MELK in neuroblastoma.

MELK-dependent EZH2 signaling has been reported to be associated with therapy resistance in adult cancers. In glioblastoma cells, both MELK and EZH2 are upregulated with radiation treatment and higher levels of both proteins are present in relapsed glioblastoma compared with diagnostic tumors (11). MELK has also been identified as a biomarker of radioresistance in breast cancer. Higher levels of MELK are detected in clinically aggressive, therapy-resistant triple-negative breast cancer and therapy-resistant triple-negative breast cancer cell lines (32). Furthermore, increased sensitivity to radiation following pharmacologic or genomic inhibition of MELK has been reported in several adult cancer models (3, 9, 10, 32). In our studies, a synergistic response was observed in neuroblastoma cells treated with OTS167 and camptothecin or radiation, suggesting that MELK-dependent EZH2 signaling inhibition also sensitizes neuroblastoma to at least these DNA-damaging agents.

Although the mechanisms responsible for the enhanced sensitivity to radiation and chemotherapy with MELK inhibition are not completely understood, EZH2 plays a recently discovered role in processing stalled replication forks into double-stand DNA breaks (DSB). This involves recruitment of the MUS81 nuclease, which generates DSB that that can serve as a substrate for homologous recombination (HR) DNA repair (36). This suggests that MELK inhibition may sensitize cells to radiation and camptothecin by interfering with the normal processing of stalled replication forks into DSB intermediates that serve as HR substrates (see model in Fig. 6E). We tested this hypothesis by treating neuroblastoma cells with either radiation or camptothecin. While radiation is commonly used to experimentally generate immediate DSBs, it also produces clustered patterns of oxidative damage of bases and sugars that can impair DNA replication (37). Consistent with this pattern of damage, radiation exposure results in secondary replication-induced DSBs that appear at relatively late time points (7–9 hours postirradiation) and are repaired by HR (38). Phosphorylation of the 32 kDa subunit of RPA32 was detected in neuroblastoma cells treated with radiation, indicative of the expected collapsed replication forks. In support of our hypothesis, OTS167 attenuated radiation-induced RPA32 phosphorylation. We additionally investigated this hypothesis using the topoisomerase I inhibitor camptothecin, which produces replication-associated DSBs that are generated by the Mus81-Eme1 endonuclease. These endonuclease-induced DSBs play an important role in dissipating the excessive supercoiling resulting from topoisomerase I inhibition, and this process ultimately aids in replication fork progression and improved cell survival (39). As expected, treatment of the neuroblastoma cells with camptothecin resulted in the formation of collapsed replication forks, again quantified as a function of phosphorylation of the 32 kDa subunit of RPA32. However, cotreatment with OTS167 antagonized the formation of collapsed replication forks after treatment with either camptothecin or radiation, supporting our proposed mechanism of action wherein MELK indirectly mediates the processing of replication-blocking lesions into DSBs that are suitable substrates for HR-mediated repair.

Taken together, our results indicate that MELK is a potential therapeutic target in neuroblastoma and provide the rationale for testing OTS167 in clinical trials for children with high-risk disease. By also inhibiting EZH2, OTS167 modifies processing of DNA lesions and increases sensitivity to radiation and camptothecin. Further studies testing the activity of OTS167 combined with other chemotherapeutic agents, radiation, and/or targeted radiopharmaceuticals such as ¹³¹I-meta-iodobenzylguanidine (MIBG) may lead to the development of novel therapeutic strategies that will prove to be effective in the treatment of patients with high-risk neuroblastoma.

J.-H. Park is a consultant/advisory board member for OncoTherapy Science, Inc. Y. Nakamura reports receiving a commercial research grant from, has ownership interest (including stock, patents, etc.) in and is a consultant/advisory board member for OncoTharapy Science. No potential conflicts of interest were disclosed by the other authors.

The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Conception and design: A. Chlenski, S.L. Cohn

Development of methodology: A. Chlenski, C. Park, M. Dobratic, H.R. Salwen, J.-H. Park, M.A. Applebaum, P.P. Connell

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Park, M. Dobratic, H.R. Salwen, B. Budke, J.-H. Park, R. Miller, E. Wilkinson, S.L. Cohn

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Chlenski, C. Park, B. Budke, M.A. Applebaum, P.P. Connell, S.L. Cohn

Writing, review, and/or revision of the manuscript: A. Chlenski, B. Budke, J.-H. Park, M.A. Applebaum, P.P. Connell, S.L. Cohn

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Dobratic, H.R. Salwen, Y. Nakamura

Study supervision: Y. Nakamura, S.L. Cohn

This work was supported in part by Hyundai Hope on Wheels, Neuroblastoma Children's Cancer Society, the Children's Neuroblastoma Cancer Foundation, the Matthew Bittker Foundation, and the Elise Anderson Neuroblastoma Research Fund (all to S.L. Cohn), Conquer Cancer Foundation (to M.A. Applebaum), Cancer Research Foundation (to M.A. Applebaum), and the Cancer Center Support Grant P30CA014599. Also supported by the NIH grants K12CA139160 (to M.A. Applebaum), K08CA226237 (to M.A. Applebaum), and 2R01CA142642 (to P.P. Connell).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.